title: Subcellular calcium measurements in mammalian cells using jellyfish photoprotein aequorin-based probes.
abstract: The jellyfish Aequorea victoria produces a 22-kDa protein named aequorin that has had an important role in the study of calcium (Ca(2+)) signaling. Aequorin reacts with Ca(2+) via oxidation of the prosthetic group, coelenterazine, which results in emission of light. This signal can be detected by using a special luminescence reader (called aequorinometer) or luminescence plate readers. Here we describe the main characteristics of aequorin as a Ca(2+) probe and how to measure Ca(2+) in different intracellular compartments of animal cells (cytosol, different mitochondrial districts, nucleus, endoplasmic reticulum (ER), Golgi apparatus, peroxisomes and subplasma-membrane cytosol), ranging from single-well analyses to high-throughput screening by transfecting animal cells using DNA vectors carrying recombinant aequorin chimeras. The use of aequorin mutants and modified versions of coelenterazione increases the range of calcium concentrations that can be recorded. Cell culture and transfection takes ∼3 d. An experiment including signal calibration and the subsequent analyses will take ∼1 d.
authors.affiliation: 1] Department of Morphology, Surgery and Experimental Medicine, Section of Pathology, Oncology and Experimental Biology, Interdisciplinary Center for the Study of Inflammation (ICSI), Laboratory for Technologies of Advanced Therapies (LTTA), University of Ferrara, Ferrara, Italy. [2].
authors.forename: Massimo
authors.initials: M
authors.lastname: Bonora
authors.forename: Carlotta
authors.initials: C
authors.lastname: Giorgi
authors.forename: Angela
authors.initials: A
authors.lastname: Bononi
authors.forename: Saverio
authors.initials: S
authors.lastname: Marchi
authors.forename: Simone
authors.initials: S
authors.lastname: Patergnani
authors.forename: Alessandro
authors.initials: A
authors.lastname: Rimessi
authors.forename: Rosario
authors.initials: R
authors.lastname: Rizzuto
authors.forename: Paolo
authors.initials: P
authors.lastname: Pinton
mesh_terms: D000331:Aequorin / Q000032:analysis* / Q000737:chemistry; D000818:Animals; D002118:Calcium / Q000737:chemistry / Q000378:metabolism*; D018929:Cell Culture Techniques; D007093:Imidazoles / Q000737:chemistry; D008163:Luminescent Measurements / Q000379:methods*; D008164:Luminescent Proteins / Q000032:analysis*; D008322:Mammals / Q000378:metabolism*; D010084:Oxidation-Reduction; D011719:Pyrazines / Q000737:chemistry; D037401:Scyphozoa / Q000378:metabolism; D014162:Transfection / Q000379:methods
chemical_list: D007093:Imidazoles; D008164:Luminescent Proteins; D011719:Pyrazines; C017144:coelenterazine; D000331:Aequorin; D002118:Calcium